SUMMARY Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.

Key Points AG-348 is a small-molecule allosteric activator of WT red cell pyruvate kinase as well as mutant enzymes associated with hemolytic anemia. Activity in vitro, in mice, and in red blood cells suggests it may address the underlying molecular pathology in PK deficiency patients.

Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase. AG-348 is an allosteric activator of the red cell isoform of pyruvate kinase (PKR) that has recently entered Phase I clinical trials in normal healthy volunteers. AG-348 increases the catalytic efficiency and enhances the protein stability of a spectrum of recombinantly expressed PKR mutant proteins that have been associated with PKD. PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the upstream glycolytic intermediate 2,3-DPG and deficiency in the PKR product adenosine triphosphate (ATP). PKR flux, e.g. the rate of carbon flow through the PKR enzyme reaction, was examined in PKD patient or wild type (WT) donor blood samples by incubation of whole blood with a stable isotope tracer, [U-13C6]-glucose. At various time points after the addition of [U-13C6]-glucose, metabolism was quenched and metabolites were extracted. Metabolite pool sizes and 13C label incorporation into glycolytic intermediates were monitored by LC/MS. The rate of label incorporation was found to be significantly slower in PKD patient red cells, consistent with decreased glycolytic activity. Treatment of PKD red cells with AG-348 ex-vivo induces changes in metabolism consistent with increased glycolytic activity including reduced 2,3-DPG levels, increased ATP levels, and increased PKR enzyme activity levels. The effect of AG-348 on red cell metabolism in vivo was evaluated in mice. C57/BL6 mice were dosed by oral gavage either with a single dose, or with multiple doses (BID) of AG-348 for 7 days. Dose levels tested were 1 mpk, 10 mpk, 50 mpk, and 150 mpk. Following the last dose, mice were bled to evaluate drug exposure and pharmacodynamic markers including 2,3-DPG and ATP levels, and PKR activity. AG-348 was demonstrated to be a well-behaved compound, with dose-proportional increase in exposure, both in the single-dose and multiple dose studies. A single dose of AG-348 resulted in a dose-dependent increase in PKR activity levels, concomitant with reduction in 2,3-DPG levels. There were no significant changes in ATP levels after a single administration of AG-348. In the multiple-dose studies, similar changes in PKR activity and 2,3-DPG levels were observed. In contrast to the single-dose study, ATP levels were observed to be robustly increased in a dose-dependent manner. The effect of AG-348 on PKR flux was assessed in whole blood from mice treated with AG-348. C57BL/6 mice were dosed by oral gavage with AG-348 (150 mg/kg twice daily [BID]) for 3 days. Whole blood was incubated with [U-13C6]-glucose and the metabolite pool sizes and rate of 13C label incorporation into glycolytic intermediates were assessed. The data were subsequently analyzed using a mathematical model to quantify flux through the PKR reaction and it was determined that AG-348 treatment significantly increased flux through the PKR reaction. Collectively, these data demonstrate that AG-348 not only potently binds to and activates the PKR enzyme in vivo, but this enzyme activation induces enhanced glycolytic pathway activity in red cells that results in profound changes in cellular metabolism, as reflected in dramatically increased ATP levels and reduced 2,3-DPG levels. As AG-348 has similar potency against the WT PKR enzyme as against tested mutant PKR enzymes in vitro, these data support the hypothesis that AG-348 treatment may similarly enhance glycolytic activity in PKD patients and thus correct the underlying pathology of PKD. Figure 1 Figure 1. Disclosures Kung: Agios Pharmaceuticals: Employment, Stockholder Other. Hill:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Jha:Agios Pharmaceuticals: Employment, Stockholder Other. Kosinski:Agios Pharmaceuticals: Employment, Stockholder Other. Clasquin:Agios Pharmaceuticals: Employment, Stockholder Other. Si:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Hixon:Agios Pharmaceuticals: Employment, Stockholder Other. Dang:A: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. Silverman:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other.

Tumors with BRCA1/2 mutations and other homologous repair deficiencies (HRD) are vulnerable to agents that target the remaining DNA repair pathways, including platinum-containing chemotherapy agents and molecules targeting poly (ADP-ribose) polymerase-1 (PARP1). Despite the clinical benefit achieved with these drugs, many patients achieve incomplete disease control and resistance often emerges. With the goal of addressing this clinical need, we applied our proprietary CRISPRomics technology to identify novel targets in cancer indications characterized by defects in DNA repair pathways. One of the top ranked targets was the deubiquitinating enzyme USP1. USP1 has established roles in DNA damage repair processes including Translesion Synthesis and the Fanconi Anemia pathway. We developed KSQ-4279, a potent, highly selective inhibitor of USP1. KSQ-4279 was active in cells, leading to the accumulation of mono-ubiquitinated substrates of USP1 and inhibited the proliferation of cancer cell lines with BRCA mutations or other HRD alterations. Studies investigating the effect of combining KSQ-4279 with PARP inhibitors revealed clear evidence of synergy in cell lines with partial or no sensitivity to each agent alone. To investigate how the distinct mechanisms of action of KSQ-4279 and PARP inhibitors would be reflected in their resistance profiles, we used our CRISPRomics technology to perform functional genomic resistance screens. The top scoring resistance genes for KSQ-4279 were distinct from those identified for PARP inhibitors, which raised the possibility that combining PARP and USP1 inhibitors may provide more durable disease control by reducing the emergence of resistance. Evaluation of KSQ-4279 in patient-derived ovarian and triple-negative breast cancer xenograft models demonstrated dose-dependent tumor growth inhibition as a single agent and in combination with PARP inhibitors. In xenograft models that were insensitive or only partially sensitive to PARP inhibitors, the combination of KSQ-4279 and Olaparib led to tumor regressions and durable tumor control. This data supports the ongoing clinical trial of KSQ-4279 in patients with tumors harboring BRCA1/2 or other HRD mutations, both as a single agent and in combination with PARP inhibitors. Citation Format: Louise Cadzow, Erica Tobin, Pamela Sullivan, Sol Shenker, Sumeet Nayak, Janid Ali, Hugh Gannon, Anne Dodson, Paula Grasberger, Alyssa Carlson, Michael McGuire, Jehrod Brenneman, Hanlan Liu, Andrew Olaharski, Kerstin Sinkevicius, Jeff Hixon, Elsa Krall, Mike Schlabach, Matt Goulet, Jeremy Wilt, Patricia Harris, Frank Stegmeier, Andrew Wylie. KSQ-4279: A first-in-class USP1 inhibitor for the treatment of cancers with homologous recombination deficiencies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND01.

Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase. Here, we describe the mechanism of action and cellular effects of AG-348, an allosteric activator of the red cell isoform of pyruvate kinase (PKR). Hundreds of mutant alleles of PKR have been identified and are known to have deleterious effects on catalytic activity, protein stability, or protein expression. We demonstrate that AG-348 can potently activate a spectrum of recombinantly expressed PKR mutant proteins, including mutations that span distinct subdomains of the enzyme. The R532W mutation is quite sensitive to AG-348 modulation, with over 4-fold activation of the enzyme activity, even as the mutation renders PKR insensitive to stimulation by its endogenous allosteric regulator fructose 1,6-bisphosphate (FBP) (Figure A). Crystallographic analysis reveals that very few mutations associated with PKD occur within the AG-348 binding pocket, accounting for its broad activity. The binding of AG-348 attenuates the thermostability defect of several mutant alleles of PKR, including the commonly observed R510Q mutant that has a half-life of ∼2% of the half-life of wild-type PKR when incubated at 53°C. Pre-incubation of the R510Q protein with AG-348 restores the half-life to ∼70% that of the wild-type enzyme (Figure B). PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the PKR substrate phosphenolpyruvate (PEP) and deficiency in the PKR product adenosine triphosphate (ATP). PKD red cells from several patients with distinct compound heterozygous PKR mutations exposed to AG-348 had increased PKR enzyme activity (up to 4-fold over control) and showed consistent dose and time-dependent metabolic responses (Figure C), including sharp reductions in PEP (up to 70% compared to control) and increases in ATP levels (up to 100% over control). These responses were observed in patient samples harboring PKR mutations that we had studied biochemically (including R486W and R510Q), but also in an instance where the mutation had not previously been biochemically characterized (A495V). In these ex-vivo settings, ATP levels in AG-348 treated cells can reach levels that are typical of normal, non-PKD red cells. These data support the hypothesis that drug intervention with AG-348 may restore glycolytic pathway activity and normalize red cell metabolism in vivo. This therapeutic approach may be an effective way to correct the underlying pathology of PKD and, importantly, provide clinical benefit to patients. Disclosures: Kung: Agios Pharmaceuticals: Employment, Equity Ownership. Hixon:Agios Pharmaceuticals: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals: Employment, Equity Ownership. Histen:Agios Pharmaceuticals: Employment, Equity Ownership. Hill:Agios Pharmaceuticals: Employment, Equity Ownership. Si:Agios Pharmaceuticals: Employment, Equity Ownership. Kernytsky:Agios Pharmaceuticals: Employment, Equity Ownership. Chen:Agios Pharmaceuticals: Employment, Equity Ownership. DeLaBarre:Agios Pharmaceuticals: Employment, Equity Ownership. Clasquin:Agios Pharmaceuticals: Employment, Equity Ownership. Ho:Agios Pharmaceuticals: Employment, Equity Ownership. Salituro:Agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:Agios Pharmaceuticals: Employment, Equity Ownership. Agresta:Agios Pharmaceuticals: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals: Employment, Equity Ownership. Dang:Agios Pharmaceuticals: Employment, Equity Ownership.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

It is well recognized that proliferating tumor cells invariably utilize aerobic glycolysis to support their high metabolic demands. Recent studies have further elucidated the paradoxical observation that this increase in glycolysis in proliferating tumor cells is often coupled with a molecular switch from the constitutively high activity PKM1 isoform to the modulated lower activity PKM2 isoform1,2, effectively constraining lower glycolysis at the last pyruvate kinase step. Here, we report an unanticipated link between glucose metabolism and amino-acid metabolic pathways in cancer cells. We characterized the biochemical and cellular effects of a small-molecule allosteric activator of the M2 isoform of pyruvate kinase (AGX-257) in various cancer cell types. We demonstrate that activation of PKM2 results in a metabolic rewiring of specific cancer cells that results in a profound dependency on the presence of the normally non-essential amino acid serine for continued cell proliferation. The induced serine auxotrophy by PKM2 activation is concomitant with reduced flux through the endogenous serine biosynthetic pathway and increased levels of high affinity serine transporters. PKM2 activators, possibly in combination with agents that modulate the cellular and extracellular serine pools, could form the basis for a new approach toward developing therapy for treating cancer. References: 1 Christofk, H. R. et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230-233 (2008). 2 Clower, C. V. et al. The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism. Proc Natl Acad Sci U S A 107, 1894-1899 (2010). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1002. doi:1538-7445.AM2012-1002